This application is a 371 of PCT/JP98/03093, filed Jul. 10, 1998.
1. Technical Field
The present invention relates to compounds having energy transfer function, and DNA sequence determination methods utilizing the compounds as a terminator, a primer or an initiator.
2. Background Art
There have been known methods for determining nucleotide sequences by preparing chain termination reaction products using a DNA polymerase or RNA polymerase, and subjecting them to separation and fractionation. Further, it has been tried to develop quicker nucleotide sequence determination methods in such ongoing projects as for elucidating gene sequences of higher animals. Some of such methods utilize a DNA fluorescence sequencer where sequencing reaction products obtained by using a fluorescence-labeled terminator are separated and fractionated by electrophoresis, the resulting DNA fragments are excited by a laser, and the emitted fluorescence is detected to determine nucleotide sequences.
It has been also known that quantum yield of fluorescent dyes can be increased based on the principle of energy transfer by using two fluorescent dyes (a donor dye and an acceptor dye). Recently, energy transfer primers have been developed which are composed of an oligonucleotide having covalently bonded two fluorescent dyes and used as a primer for sequencing reactions based on the principle of energy transfer [see, for example, Nature Medicine, 2, 246-249 (1996), WO95/21266, Japanese Patent Unexamined Publication (KOKAI) No. Hei 10-88124/1998 etc.].
In order to realize faster DNA sequencing, systems capable of analyzing multiple samples simultaneously have been developed. In particular, the capillary fluorescence sequencer has an ideal structure, in which loading of samples can be easily automated, and multiple capillaries can be easily used without crossing of lanes. For this fluorescence sequencer, two kinds of optical systems have been used in order to realize the use of larger number of capillaries.
One is the scanning method, and the other is the imaging method. For the both cases, it is essential for the use of larger number of capillaries that a highly sensitive detector, which can detect even a small amount of DNA, should be available. In the scanning method, in order to finish the sequencing within a certain period of time regardless of the number of capillaries, time assigned to each capillary should be shorter if the number of capillaries became larger. Therefore, a detector of higher sensitivity is required. In the imaging method, the range of the field covered by a detector is constant regardless of the number of capillaries. In order to use a larger number of capillaries, it is necessary to increase the number of picture elements of optics element, and decrease the diameter of each capillary. Therefore, it also requires a detector having higher sensitivity.
On the other hand, as discussed above, the technique for utilizing the fluorescence primer based on the principle of energy transfer for DNA sequencing has been developed in order to increase DNA detection sensitivity. However, because the technique utilizes the fluorescence primer, it requires complex operation procedure of which termination reaction should be performed for each of the four kinds of bases, A, G, C and T, and the mixed sequencing reaction products should be subjected to electrophoresis. In addition, because the sequencing based on the transcription reaction by a promoter-dependent RNA polymerase does not use a primer, the energy transfer primer cannot be used in it.
In order to realize faster DNA sequencing, it is necessary to use a highly sensitive detection system with high quantum yield such as energy transfer in a multiple capillary sequencer. On the contrary, however, the systems utilizing the energy transfer primers suffers from technical limitations such as complicated pretreatment (DNA polymerase system) and necessity of primer itself (RNA polymerase system).
Therefore, an object of the present invention is to provide a means which eliminates the drawbacks of the systems utilizing the energy transfer primers, i.e., which is adaptable to the transcription reaction systems utilizing RNA polymerases, and enables sequencing methods capable of high sensitivity detection utilizing the energy transfer and not requiring the complicated procedure consisting of mixing of the four kinds of reaction products for A, G, C and T separately obtained in the preliminary processes.
In particular, an object of the present invention is to provide a compound which can utilize the principle of energy transfer to afford high sensitivity, and a method for determining nucleotide sequences of DNA which utilizes such a compound as mentioned above as a terminator, and can detect labeled DNA fragments with high sensitivity based on the chain terminator method.
Meanwhile, the energy transfer primer disclosed in WO95/21266 is composed of two reporters causing the energy transfer which are connected with a part of oligonucleotide constituting the primer used as a linker. However, such a primer is practically disadvantageous, because a distinct primer must be synthesized for each of oligonucleotides used as primers having different sequences. On the other hand, the energy transfer primer of Japanese Patent Unexamined Publication (KOKAI) No. Hei 10-88124/1998 is composed of two reporters connected with an aliphatic or aromatic residue used as a linker. Therefore, it does not suffer from the problem observed in the primer of WO95/21266, but it may suffers from another problem that, when a longer linker is desired to realize a longer distance between the two reporters, it may be difficult to obtain a desired distance between the two reporters because of the bonding scheme of the linker even though the linker has a long chain.
Therefore, another object of the present invention is to provide a compound useful as the energy transfer primer which solves the above problem, i.e., which does not use a part of a primer sequence as the linker and enables easy control of the distance between the two reporters.
A further object of the present invention is to provide a method for determining nucleotide sequences of DNA which uses the above compound as the energy transfer primer.
In a method for determining nucleotide sequences using a terminator, an RNA polymerase such as T7 RNA polymerase is used for a reaction in a mixture of ribonucleoside 5xe2x80x2-triphosphates and 3xe2x80x2-deoxyribonucleotides. In this reaction, ribonucleotides and 3xe2x80x2-deoxyribonucleotides having a base corresponding to the sequence of the template are sequentially incorporated into a ribonucleotide sequence to synthesize a polyribonucleotide sequence. The resulting polyribonucleotides (nucleic acid transcription products) are then separated, and nucleic acid sequence is read from the resulting separated fractions to determine the nucleotide sequence of the DNA. For example, fluorescence-labeled 3xe2x80x2-dNTP derivatives are used as the terminators of the nucleic acid transcription for the nucleotide sequence determination.
However, terminators composed of 3xe2x80x2-dNTP having various kinds of labels may be difficult to be incorporated into a nucleic acid sequence, depending on the kind of the labels and the bonding scheme of the labels. In particular, when the chain length becomes longer, such a tendency becomes more serious. To deal with this problem, the nucleotide sequence may also be determined by using unlabeled compounds as the terminators and labeled initiators. Also in such a case, sensitivity of the label is important like in the labeled terminators mentioned above.
Therefore, a further object of the present invention is to provide a compound having energy transfer function which can be used as an initiator (transcription initiator) in a method for determining nucleotide sequences of DNA using an RNA polymerase without using a labeled terminator for the nucleotide sequence determination.
A still further object of the present invention is to provide a method for determining DNA sequences utilizing the above compound as an energy transfer initiator.
The present invention relates to a compound represented by the following general formula (1): 
wherein Q represents a mono- or oligonucleotide residue, V represents xe2x80x94Cxe2x89xa1Cxe2x80x94(CH2)n1xe2x80x94NHxe2x80x94 or xe2x80x94CHxe2x95x90CHxe2x80x94(CH2)n2xe2x80x94NHxe2x80x94 wherein n1 and n2 represent an integer not less than 1, R1 represents a trivalent group, R2 and R3 independently represent hydrogen atom or a hydrocarbon residue, or R2 and R3 may join to form a ring together with the adjacent CH and NH, W1 and W2 independently represent a fluorescent group, and m represents an integer not less than 1.
In the above compound, R2, R3 and m are preferably selected so that the distance between W1 and W2 should be within the range of 10-100 xc3x85.
In the above compound, Q is preferably a 2xe2x80x2, 3xe2x80x2-dideoxyribonucleotide residue or a 3xe2x80x2-deoxyribonucleotide residue. Such a compound can be used as a terminator for the DNA sequence determination method based on the chain terminator method.
The present invention relates to a method for determining DNA sequences based on the chain terminator method, characterized in that the chain termination reaction is performed by using a compound represented by the general formula (1) where Q is a 2xe2x80x2,3xe2x80x2-dideoxyribonucleotide residue or a 3xe2x80x2-deoxyribonucleotide residue as a terminator.
This method includes a method using DNA polymerase and a compound represented by the general formula (1) where Q is a 2xe2x80x2,3xe2x80x2-dideoxyribonucleotide residue as the terminator, and a method using RNA polymerase and a compound represented by the general formula (1) where Q is a 3xe2x80x2-deoxyribonucleotide residue as the terminator.
In this method, four kinds of compounds corresponding to four kinds of bases are used as terminators, wherein the compounds are selected from the compounds represented by the general formula (1) and each of which has one of different four kinds of fluorescent groups as at least one of W1 and W2, and the chain termination reaction using the above four kinds of compounds can be performed in the same reaction system.
The present invention relates to a compound represented by the general formula (1) where Q is an oligonucleotide residue having a 2xe2x80x2-deoxyribonucleotide residue at its end. This compound can be used as a primer in the DNA sequence determination method based on the primer method.
The present invention further relates to a method for determining DNA sequences based on the primer method, characterized in that the above compound is used as a primer. In this method, an unlabeled terminator can be used.
The present invention relates to a compound represented by the general formula (1) where Q is a mono- or oligonucleotide residue not having a phosphate group, or having a mono- or diphosphate group at the 5xe2x80x2 end. This compound can be used as an initiator in DNA sequence determination methods based on the chain terminator method.
The present invention relates to a method for determining DNA sequences based on the chain terminator method, characterized in that the chain termination reaction is performed by using an initiator comprising a mono- or oligonucleotide residue not having a phosphate group, or having a mono- or diphosphate group at the 5xe2x80x2 end and two kinds of reporters that can be a donor and an acceptor of energy transfer, and an RNA polymerase.
In this method, the compound represented by the general formula (1) where Q is a mono- or oligonucleotide residue not having a phosphate group, or having a mono- or diphosphate group at the 5xe2x80x2 end can be used as an initiator. As the terminator, an unlabeled terminator can be used.
The present invention further relates to a method for determining DNA sequences based on the chain terminator method characterized in that the chain termination reaction is performed by using a terminator comprising a 3xe2x80x2-deoxyribonucleotide residue and two kinds of reporters that can be a donor and an acceptor of energy transfer, and an RNA polymerase.
In this method, two reporters on the terminator are preferably arranged with a distance sufficient for causing energy transfer from the donor to the acceptor. The distance sufficient for causing energy transfer from the donor to the acceptor is, for example, in the range of 10-100 xc3x85. The reporters are, for example, selected from the group consisting of fluorescent groups, phosphorescent groups, spin-labeled groups and groups having high electron density.
In this method, for example, the donor contained in the terminator is selected from the group consisting of fluorescein dyes, rhodamine dyes and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes, and the acceptor is selected from the group consisting of fluorescein dyes, rhodamine dyes and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes.
In this method, four kinds of terminators corresponding to the four kinds of bases are used as the acceptor, provided that each of which terminators has one of different four kinds of reporters. And the chain termination reaction with the above four kinds of terminators can be performed in the same reaction system.
In the above nucleotide sequence determination methods of the present invention, the chain termination reaction is preferably performed in the presence of inorganic pyrophosphatase.